Glycoproteins are widespread in nature and have been implicated in many disease processes. For example, in atherosclerosis alterations in glycoprotein content and structure have been reported while in diabetic microangiopathy they are changes in the basement membrane. Probably these changes reflect alterations in the pathway of synthesis and perhaps involve disturbances in the regulation mechanisms. Thus, it is important to understand how glycoproteins are synthesized in normal cells so that these pathways can be compared in the diseased state. Many of these glycoproteins are on the N-glycosidic type and have a core oligosaccharide of manose and GlcNAc attached to protein in a glycosaminyl-asparagine linkage. Included in this group are many secretory proteins such as the serum lipoproteins, blood clotting proteins and so on. In recent years it has become clear that the core oligosaccharide of many N-glycosidically linked glycoproteins is synthesized in a novel fashion involving lipid-linked saccharide intermediates. However, while the general pathway is clear there are many gaps in our understanding such as the individual steps in the assembly process, in where these reactions occur, in how the pathway is regulated and in how the polyisoprenol lipids themselves are synthesized. We plan to examine these questions using an enzyme system which forms a whole series of lipid-linked oligosaccharides from GDP-(14C)mannose or from UDP-(3H)GlcNAc in the presence of GDP-mannose. Since the oligosaccharides differ from each other only by the presence of an additional sugar residue, this system offers the advantages of being able to examine the stepwise synthesis of these compounds. We can also examine changes in individual activities as influenced by various lipid substrates or inhibitors. Since we have some evidence that the carrier lipid for GlcNAc is different from that for mannose, we will try to isolate and characterize this lipid since this may be an important control point in the lipid-linked saccharide pathway. We will also attempt to solubilize and study the glycosyl transferases which are involved in this pathway. Transfer of the oligosaccharide to protein will be studied in cell-free extracts to compare the rate of transfer of the various sized lipid-linked oligosaccharides. In vitro transfer to nascent chains will also be examined as will the nature of the final glyccoprotein product.